This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ubiquitin signaling is an important mechanism is nearly every cellular process. E3 ubiquitin ligases specify the substrate and catalyze the transfer from an E2 ubiquitin conjugating enzyme to that protein. Though E3 enzymes are a large and well-studied class of proteins, little is known about how the E3 catalyzes this transfer. We plan to interrogate the functional consequences of mutation at every amino acid of an E3 ligase. To this end, we have chosen to study the U-box domain of UBE4B and constructed a library of 1 million UBE4B mutants displayed on the coat of T7 phage. We use the UBE4B phage in in vitro ubiquitination reactions to test their E3 activity. With the addition of E1 and E2 enzymes, the UBE4B catalyzes autoubiquitination using Flag-tagged ubiquitin. This procedure allows us to select for enzymatically active UBE4B-phage by incubation with anti-Flag beads. Nonspecifically bound phage are washed away and bound phage are eluted by competition with Flag peptide. The eluted phage are amplified and subjected to more rounds of selection. Using high throughput sequencing to determine genotypes in the input pool of phage versus selected phages, we can track how each mutation performs during the selection experiments. This experiment will give us insight into the function of every amino acid in the U-box domain of UBE4B and may reveal the functional elements of E3 catalysis.